Isolation and Culture of Rat Aortic SMCs

Rat aortic SMCs were isolated and cultured as described previously.^{20 (link)} Briefly, rat aortas were excised and dissected free of adventitia and fat. The vessels were further digested by collagenase type I (Worthington Biochemical, 1 mg/ml) and elastase type III (sigma 0.125 mg/ml) at 37°C in a CO₂ humidified chamber. Then, cell suspensions were centrifuged and the cell pellets were re suspended and grown in DMEM with 10% fetal bovine serum and penicillin and streptomycin (1%) in 5% CO₂ at 37°C. The SMC phenotype was determined as described above. Cells of passages 2–3 were used for the short interfering (si)RNA transfection study. SMCs were transfected with control siRNA or siRNA targeting rat calpain-2 or filamin A (Ambion - Silencer Select validated siRNA, ThermoFisher Scientific) sequences using RNAiMax lipofectamine transfection reagent (ThermoFisher Scientific). After 48 h of transfection, cells were treated with either vehicle or AngII (100 nM) for 24 h and then harvested for western blot analyses.